The a7 neuronal nicotinic acetylcholine receptor is an important therapeutic target, relevant to Alzheimer's Disease, stroke, and schizophrenia. Understanding how this receptor responds to natural and experimental agonists is crucial for the therapeutic targeting of a7. We have shown that stimulation of a7 receptors by relatively low agonist concentrations can support an equilibration between activation and desensitization, resulting in a functional modality that we hypothesize to be the basis for therapeutic effects. In this continuation we will study how a7-selective agonists both activate this receptor and, in some cases, produce down-regulation of function through secondary inhibitory or desensitizing effects. Our experiments utilize structurally diverse a7-selective agonists including large molecules such as benzylidene anabaseines (BA) and indole tropanes (IT) and smaller probes for receptor selectivity such as choline and tropane. Our data suggest that BA compounds with residual inhibitory effects interact with hydrophobic residues on the margins of the ACh binding site. We will test that hypothesis through the use of site-directed mutants and new experimental agonists. We will develop novel compounds including additional indole-tropanes (IT) to evaluate as agonists and use as experimental probes. The prototype for the IT compounds is tropisetron, a 5HT3 receptor antagonist and a7-selective agonist. Our preliminary studies show that while the indole moiety in tropisetron has activity with 5HT3 receptors, the tropane group is, on its own, an a7 agonist. Indole conjugation appears to make tropisetron a more potent, though less efficacious, agonist for a7 than the small agonist tropinone. We hypothesize that, based either on intramolecular effects or specific interactions between the drug and amino acids in or near the agonist binding site, substitutions of the indole will modify the properties of IT agonists in ways that will be predictable based on our previous studies of BA compounds and models of the receptor binding site. The structurally diverse BA and IT agonists are much larger molecules than the endogenous agonists, ACh and choline, and are likely to have binding sites that include more points of contact on the receptor than the binding site of ACh. We will conduct scanning cysteine accessibility experiments to identify the binding sites for large and small molecular probes, utilizing both selective and nonselective agonists as well as competitive antagonists. We will define both the ligand and protein basis through which certain drugs achieve selectivity for a7 receptors. Taken together, our studies will define effective ways to activate a7 receptors with potentially therapeutic agents and will also provide insights into how new agents may be designed to selectively activate this receptor with minimized inhibitory side-effects due to receptor desensitization or the non-selective activation of other receptor subtypes.